Unit 2.

Separation and Purification

Techniques of Biomolecules
Prepared by: Alexis M. Labrador UST Department of Biochemistry – Faculty of Pharmacy
 Selection of Starting Material: CELLS

Biological Samples extracted from microorganisms, plants, animals and humans:

 Structure and Diversity of Cells

ALL cells have a Plasma membrane that separates the cell contents from the
extracellular environment. It has Phospholipid bilayer, which is an amphipathic
molecules, where membrane proteins are attached in place as domains spanning a
hydrophobic core.
 Structure and Diversity of Cells

Cells – Prokaryote or Eukaryote?

- from animals, plants, microbes or humans
Considerations in Sample Preparations:

Cells which are not attached to others (such

as blood or suspension tissue cultures) can
be separated if they have distinct shapes,
densities or characteristics which can be
marked (such as charge, antigen or enzyme
presence).

Cells which are part of a more solid tissue

(such as liver or kidney) will first need to be
separated from all connections with other
cells.
Considerations in Sample Preparations:

Factors that affect sample preparation:

1. Source of biological specimen or sample
type
2. Chemical and structural heterogeneity of
desired molecules
3. Cellular or subcellular location of
biomolecule of interest
4. Required Actual yield
5. Proposed downstream application
 Cell Lysis or Homogenization

Cell homogenization – is the process of breaking or lysing the cells gently to release
the cell contents, particularly organelles and biomolecules, for subsequent analysis.

Cell fractionation – the subsequent isolation of organelles and subsequent purification

of target biomolecules.
Cell Lysis or Homogenization
Characteristics of Traditional Methods of Cell lysis:

• It may require expensive equipment.

• It may be cumbersome to use.

• Reproducibility may vary.

• Mechanical methods are generally not compatible with high-throughput and

small volumes.

• Protein denaturation and aggregation can occur.

• Cells disrupt at different times, so subcellular components may be subjected

to ongoing disruptive forces.
 Traditional Methods of Cell Lysis

DISADVANTAGES:
1. Localized heating can occur within the sample leading to protein denaturation and
aggregation.
2. Reproducibility with homogenization and grinding methods can be challenging due
to differing sample handling techniques.
3. Cells disrupt at different times where the contents are subjected to disruptive
forces.
4. Some physical disruption methods require fairly expensive equipment, such as the
French press and sonicator.
 TM-based Cell Lysis: Physical-Disruption

1. Mechanical disruption – rely on the use of rotating blades to grind and disperse
cells and tissues
Apparatus: Waring Blender and Polytron
 TM-based Cell Lysis: Physical-Disruption

2. Liquid homogenization - most widely used cell disruption technique for small
volumes and cultured cells which are sheared by forcing them through a narrow
space. There are three common techniques namely:

1. Dounce homogenizer 2. Potter-Elvehjem homogenizer 3. The French Press

TM-based Cell Lysis: Physical-Disruption

3. Sonication - The method uses pulsed, high frequency sound waves to

agitate and lyse cells, bacteria, spores and finely diced tissue.

Apparatus: Sonicator
 TM-based Cell Lysis: Physical-Disruption

4. Freeze-Thaw - is commonly used to lyse

bacterial and mammalian cells. The technique
involves freezing a cell suspension in a dry
ice/ethanol bath or freezer and then thawing
the material at room temperature or 37°C.

Recommended for lysis of mammalian cells in

some protocols.
 TM-based Cell Lysis: Physical-Disruption

5. Manual grinding - is the most common method used to disrupt plant cells.
Tissue is frozen in liquid nitrogen and then crushed using a mortar and pestle.
 Additives to Physical Disruption

1. Hypotonic buffer – causes the cells to swell and readily burst under physical
shearing.
 Additives to Physical Disruption

2. Lysozyme - used to digest the

polysaccharide component of yeast and
bacterial cell walls. Alternatively,
processing can be expedited by treating
cells with glass beads in order to facilitate
the crushing of cell walls.
Characteristics of Reagent-based Cell lysis methods:

• It is a rapid, gentle, efficient, and reproducible method, and leads to a high

protein yield.

• It can be used to extract total protein or subcellular fractions or organelles from

various sample types.

• It works by disrupting the lipid membrane and/or cell wall.

• Some components may need to be removed for downstream analysis.

• High concentrations of salts and detergents are not compatible with protein
assays and mass spectrometry.
 Reagent-based Cell lysis methods

• Cell lysis solutions are detergent-based, buffers and reagent sets that have been
optimized for particular cell lysis applications.
• These methods of disruption of cells utilize osmotic pressure or detergent
interactions to destroy cells’ walls and membranes. They are also efficient for
homogenization of nuclear and mitochondrial membranes in cell extracts.
 Reagent-based Cell lysis methods

Detergents for Cell lysis – a milder

and easier alternative method of cell
disruption that is used in conjunction
with homogenization and mechanical
grinding. Detergents break the lipid
barrier surrounding cells by
solubilizing proteins and disrupting
lipid–lipid, protein–protein and
protein–lipid interactions.

Ex: Ionic detergents

Non-ionic (Triton X-100, NP40)
Zwitterionic (CHAPS)
 Reagent-based Cell lysis methods

Detergents for Cell Lysis:

1. Ionic detergents - are strong solubilizing agents and tend to denature
proteins, thereby destroying protein activity and function.
2. Non-ionic detergents – include Triton X-100 and NP-40. These are
milder and less denaturing detergents which are used to solubilize
membrane proteins that is critical to maintain protein function and/or
retain native protein–protein interactions for downstream applications.
3. Zwitterionic detergents (CHAPS) – CHAPS is a sulfobetaine derivative
of cholic acid. This zwitterionic detergent is useful for membrane protein
solubilization when it is important to maintain protein activity.
 Reagent-based Cell lysis methods

Important considerations for choosing detergent for cell lysis:

1. Sample type whether it is derived from animals, plants or bacteria
2. Buffer
3. pH of the cell lysis solution
4. Salt concentration
Mammalian Cell Lysis Reagents

RIPA (Radioimmunoprecipitation) buffer -

popular choice for lysis and protein extraction of
mammalian cells or soft tissue. The formulation includes
two ionic detergents and one nonionic detergent in Tris
buffer: 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP40,
1% sodium deoxycholate and 0.1% sodium dodecyl sulfate
(SDS).
 Mammalian Cell Lysis Reagents

MPER (Mammalian Protein Extraction Reagent) - was developed as an effective

yet milder alternative to RIPA buffer which uses a non-denaturing detergent to
prepare total cell lysate that is compatible with many downstream assays.
 Mammalian Cell Lysis Reagents

TPER (Tissue Protein

Extraction Reagent) - designed
for total protein extraction from
mammalian tissue samples,
including heart, liver, kidney and
brain. It utilizes a non-denaturing
detergent in 25 mM bicine, 150 mM
NaCl (pH 7.6), and is used in
conjunction with mechanical or
manual homogenization.
 Bacterial Protein Extraction reagents

B-PER Bacterial Protein

Extraction Reagents gently lyse E.
coli and other species of bacterial
cells and effectively extract soluble
native and recombinant proteins.
Can be used for Gram-negative
bacteria, S. aureus, H. pylori and E.
coli strains BL21(D3), JM109, DH5a
and M15.
 Insect, yeast and plant cell lysis reagents

Y-PER Yeast Protein Extraction Reagent penetrates the tough yeast cell wall,
perforating the cell wall and membrane and extracting soluble protein without
completely damaging overall cell structure.

Two formulations:

1. YPER – high salt (> 300mM) and is effective for S. cerevisiae, S. pombe, C.
albicans and P. pastoris (as well as various Gram-positive and Gram-negative
bacteria).

2. YPER Plus Dialyzable Yeast Protein Extraction Reagent - is a phosphate-free,

low-salt formulation with a dialyzable detergent. It is validated for use primarily
with S. cerevisiae.
 Insect, yeast and plant cell lysis reagents

I-PER Insect Cell Protein Extraction

Reagent enables gentle extraction of
soluble protein from baculovirus-
infected insect cells grown in
suspension of monolayer culture (both
Sf9 and Sf21 cells). The reagent
maintains functionality of extracted
proteins and is directly compatible with
downstream applications.
 Insect, yeast and plant cell lysis reagents

P-PER Plant Protein Extraction

Reagent includes an organic lysing
reagent and two aqueous reagent that is
effective in extracting plant protein. The
method has been validated for use with
multiple plan organs (leaf, stem, root,
seed and flowers); multiple plant species
(Arabidopsis, tobacco, maize, soybeans,
peas, rice and spinach); and fresh, frozen
and dehydrated tissue sources.
Additives for Reagent-based Cell lysis methods

1. Protease and phosphatase inhibitors – these are small molecules or

compounds added to the lysis buffer that inactive or block the activities of
proteases and phosphatases.
2. Nucleases – organic compounds capable of inhibiting the degradative
actions of DNase for DNA and RNase for RNA.
3. Lysozyme solution – mixture of compounds added to lysis buffer for the
purpose of additive enzyme that helps the disintegration of polysaccharide
layer and peptidoglycan layers in bacterial cells.
 Important considerations in cell
homogenization conditions:

 Low temperature (4 °C)

 Buffer with pH ranging from 7.0 to 7.4 and suitable salt concentration
 High Osmolarity (15% sucrose) – Isotonic condition
 Addition of specific ions (CaCl2, MgCl2) for stabilization
 Chelating agent (EDTA) for protease inhibition
 Reducing agent (mercaptoethanol) – prevents damaging oxidation
reactions
 Renaturation buffers to fold back proteins into their 3D conformation
Example of Renaturation buffers
 Dialysis, Ultrafiltration and Lyophilization

PRINCIPLE INVOLVED:
Diffusion is the random,
thermal movement of
molecules in solution that
leads to the net movement of
molecules from an area of
higher concentration to a
lower concentration until
equilibrium is reached.
 DIALYSIS

1. DIALYSIS – one of the oldest

technique for separation of
biomolecules on the basis of their
molecular size.
Involves the use of
semipermeable membrane that
contains the biochemical solutions,
which is placed in low-ionic
strength buffer for separation.
 DIALYSIS

Processes in Dialysis:
1. Only small molecules diffuse through the semi
permeable membrane
2. At equilibrium, the concentration of small
molecules is the same inside and outside of
the membrane.
3. Macromolecules remain in the bag.
DIALYSIS

TWO Variables in the method of Dialysis:

1. The type of membrane – it can be collodion, cellophane and cellulose

2. The size of the pores or the molecular weight cutoff.

Only molecules or ions smaller than the molecular weight will
move out of the dialysis bag.
Applications and Limitations of Dialysis

1. Removal of salts and low molecular weight compounds.

2. Buffer exchange.

3. Concentration of macromolecules.

4. Purification of biotechnological products.

5. Medical applications: kidney dialysis and hemodialysis

Advantages of Dialysis

1. Dialysis is still in use today for it is very simple and is still the only way to
deal with large-volume samples.
2. Characterization of a candidate drug in serum binding assays or detailed
study of antigen-antibody interactions.
3. Proves to be the most accurate method available.
4. inexpensive and easy to perform.

Disadvantage: Slow process several hours for completion, and thus, has
been replaced by gel filtration for most applications. Other forms of dialysis
includes flow-dialysis and pressure-dialysis.
 ULTRAFILTRATION

Principle: Ultrafiltration operates according to diffusion under pressure.

Involves the separation of molecular species on the basis of size, shape and/or
charge.

Serves 2 purposes:
1. Purification
2. Concentration
 ULTRAFILTRATION

Factors in choosing Membranes for Ultrafiltration

1. Optimum flow rate
2. Molecular specificity
3. Molecular weight cutoff

2 applications of membrane filtration:

1. Desalting buffers or other solutions
2. Clarification of turbid solutions by removal of micron- or submicron-sized
particles.
 ULTRAFILTRATION MEMBRANES

• Typically have molecular weight cutoffs in the range of 100 to 1,000,000.

• Usually composed of 2 layers: (1) a thin, 0.1-0.5 μm, surface, semipermeable
membrane made from either of the following materials: cellulose acetate,
nylon, polyvinylidene and cellulose nitrate and (2) thicker, inert, support base.
• Membrane filters can be customized according to desired pore size ranging
from 0.025 to 15 μm.
• Filters require suction, pressure, or centrifugal force for liquid to flow. Example
for 0.45 μm membrane – 57mL/min cm2 at 10 psi.
Electron micrograph of an ultrafiltration membrane
 ULTRAFILTRATION DEVICES

1. Macro separations – generally up to 50L or for solutions larger than a few

milliliters. It requires the use of gas-pressurized cells or suction-filter devices
for the liquid to flow.
2. Micro separations – milli- to microliter range, which commonly uses
disposable devices. These devices offer user simplicity, time saving, and high
recovery.